The present invention is directed to novel Borrelia burgdorferi polypeptides and uses thereof.
Lyme borreliosis (Lyme disease) is the most common vector-borne infection in the United States [S. W. Barthold, et al., xe2x80x9cAn Animal Model For Lyme Arthritisxe2x80x9d, Ann. N.Y. Acad. Sci., 539, pp. 264-73 (1988)]. It has been reported in every continent except Antarctica. The clinical hallmark of Lyme Disease is an early expanding skin lesion known as erythema migrans, which may be followed weeks to months later by neurologic, cardiac, and joint abnormalities.
The causative agent of Lyme disease is a spirochete known as Borrelia burgdorferi, transmitted primarily by Ixodes ticks of the Ixodes ricinus complex. B. burgdorferi has also been shown to be carried in other species of ticks and in mosquitoes and deer flies, but it appears that only ticks of the I. ricinus complex are able to transmit the disease to humans.
Lyme disease generally occurs in three stages. Stage one involves localized skin lesions (erythema migrans) from which the spirochete is cultured more readily than at any other time during infection [B. W. Berger et al., xe2x80x9cIsolation And Characterization Of The Lyme Disease Spirochete From The Skin Of Patients With Erythema Chronicum Migransxe2x80x9d, J. Am. Acad. Dermatol., 3, pp. 444-49 (1985)]. Flu-like or meningitis-like symptoms are common at this time. Stage two occurs within days or weeks, and involves spread of the spirochete through the patient""s blood or lymph to many different sites in the body including the brain and joints. Varied symptoms of this disseminated infection occur in the skin, nervous system, and musculoskeletal system, although they are typically intermittent. Stage three, or late infection, is defined as persistent infection, and can be severely disabling. Chronic arthritis, and syndromes of the central and peripheral nervous system appear during this stage, as a result of the ongoing infection and perhaps a resulting auto-immune disease [R. Martin et al., xe2x80x9cBorrelia burgdorferi-Specific And Autoreactive T-Cell Lines From Cerebrospinal Fluid In Lyme Radiculomyelitisxe2x80x9d, Ann Neurol., 24, pp. 509-16 (1988); D. Gross et al., xe2x80x9cIdentification of LFA-1 as a Candidate Autoantigen in Treatment-Resistant Lyme Arthritisxe2x80x9d, Science 281, pp. 703-706 (1998)].
B. burgdorferi is much easier to culture from the tick than from humans, therefore at present, Lyme disease is diagnosed primarily by serology. The enzyme-linked immunosorbent assay (ELISA) is one method of detection, using sonicated whole spirochetes as the antigen [J. E. Craft et al., xe2x80x9cThe Antibody Response In Lyme Disease: Evaluation Of Diagnostic Testsxe2x80x9d, J. Infect. Dis., 149, pp. 789-95 (1984)]. However, false negative and, more commonly, false positive results are associated with currently available tests.
At present, all stages of Lyme disease are treated with antibiotics. Treatment of early disease is usually effective, however the cardiac, arthritic, and nervous system disorders associated with the later stages often do not respond to therapy [A. C. Steere, xe2x80x9cLyme Diseasexe2x80x9d, New Eng. J. Med., 321, pp. 586-96 (1989)].
Like Treponema pallidum, which causes syphilis, and leptospirae, which cause an infectious jaundice, Borrelia belong to the eubacterial phylum of spirochetes [A. G. Barbour and S. F. Hayes, xe2x80x9cBiology of Borrelia Speciesxe2x80x9d, Microbiol. Rev., 50, pp. 381-400 (1986)]. Borrelia burgdorferi have a protoplasmic cylinder that is surrounded by a cell membrane, then by flagella, and then by an outer membrane.
The B. burgdorferi outer surface proteins identified to date are believed to be lipoproteins, as demonstrated by labeling with [3H]palmitate [M. E. Brandt et al., xe2x80x9cImmunogenic Integral membrane Proteins of Borrelia burgdorferi Are Lipoproteinsxe2x80x9d, Infect. Immun., 58, pp. 983-91 (1990)]. The two major outer surface proteins are the 31 kd outer-surface protein A (OspA) and the 34 kd outer surface protein B (OspB). Both proteins have been shown to vary from different isolates or from different passages of the same isolate as determined by their molecular weights and reactivity with monoclonal antibodies. OspC is a 22 kDa membrane lipoprotein previously identified as pC [R. Fuchs et al., xe2x80x9cMolecular Analysis and Expression of a Borrelia burgdorferi Gene Encoding a 22 kDa Protein (pC) in Escherichia colixe2x80x9d, Mol. Microbiol., 6, pp. 503-09 (1992)]. OspD is said to be preferentially expressed by low-passage, virulent strains of B. burgdorferi B31 [S. J. Norris et al., xe2x80x9cLow-Passage-Associated Proteins of Borrelia burgdorferi B31: Characterization and Molecular Cloning of OspD, A Surfaced-Exposed, Plasmid-Encoded Lipoproteinxe2x80x9d, Infect. Immun., 60, pp. 4662-4672 (1992)].
Additional B. burgdorferi proteins identified to date include the 41 kD flagellin protein, which is known to contain regions of homology with other bacterial flagellins [G. S. Gassman et al., xe2x80x9cAnalysis of the Borrelia burgdorferi GeHo fla Gene and Antigenic Characterization of Its Gene Productxe2x80x9d, J. Bacteriol., 173, pp. 1452-59 (1991)] and a 93 kDa protein said to be localized to the periplasmic space [D. J. Volkman et al., xe2x80x9cCharacterization of an Immunoreactive 93 kDa Core Protein of Borrelia burgdorferi With a Human IgG Monoclonal Antibodyxe2x80x9d, J. Immun., 146, pp. 3177-82 (1991)].
Recently, immunization of mice with recombinant OspA has been shown to be effective to confer long-lasting protection against subsequent infection with B. burgdorferi [E. Fikrig et al., xe2x80x9cLong-Term Protection of Mice from Lyme Disease by Vaccination with OspAxe2x80x9d, Infec. Immun., 60, pp. 773-77 (1992)]. Protection by the OspA immunogens used to date appears to be somewhat strain specific, probably due to the heterogeneity of the OspA gene among different B. burgdorferi isolates. For example, immunization with OspA from B. burgdorferi strain N40 confers protection against subsequent infection with strains N40, B31 and CD 16, but not against strain 25015 [E. Fikrig et al., xe2x80x9cBorrelia burgdorferi Strain 25015: Characterization of Outer Surface Protein A and Vaccination Against Infectionxe2x80x9d, J. Immun., 148, pp. 2256-60 (1992)].
An additional problem with OspA as a protective immunogen is cross-reactivity at the T cell level observed between OspA165-173 and LFA-1xcex1. [D. Gross et al., xe2x80x9cIdentification of LFA-1 as a Candidate Autoantigen in Treatment-Resistant Lyme Arthritisxe2x80x9d, Science 281, pp. 703-706 (1998)]. As described above, a prominent late manifestation of infection with B. burgdorferi is Lyme arthritis[A. C. Steere, et al., Ann. Int. Med 90: 896 (1979); A. C. Steere, et al., Ann. Int. Med. 107: 725 (1987); A. C. Steere, et al., N. Eng. J. Med. 321: 586 (1989); A. C. Steere, et al., N. Eng. J. Med. 323: 219 (1990); A. C. Steere, et al., Arthritis Rheum. 37: 878 (1994)]. About 10% of patients with Lyme arthritis develop a condition known as antibiotic-resistant Lyme arthritis, which typically affects one knee for months to years after multiple courses of antibiotics [A. C. Steere, et al., Ann. Int. Med. 90: 896 (1979); A. C. Steere, et al., Ann. Int. Med. 107: 725 (1987); A. C. Steere, et al., N. Eng. J. Med. 321: 586 (1989); A. C. Steere, et al., Arthritis Rheum. 37: 878 (1994)]. Such patients have no detectable spirochetal DNA in joint fluid after antibiotic therapy, which suggests that the spirochete has been eliminated by this treatment [J. F. Bradley, et al., Ann. Int. Med. 120: 487 (1994); J. J. Nocton et al., N. Eng. J. Med. 330: 229 (1994)]. The increased frequency of the HLA-DRB1*0401 allele in these patients suggests an autoimmune etiology [unpublished data] (Kalish et al., 1993 Infect Immun 61:2774-9).
Recent work has identified LFA-1xcex1 as a candidate autoantigen in treatment-resistant Lyme arthritis [D. Gross et al., xe2x80x9cIdentification of LFA-1 as a Candidate Autoantigen in Treatment-Resistant Lyme Arthritisxe2x80x9d, Science 281, pp. 703-706 (1998)]. The immunodominant epitope of OspA for T helper cells was identified, and a homology search revealed a peptide from human leukocyte function-associated antigen 1 (LFA-1xcex1). Individuals with treatment-resistant Lyme arthritis, but not other forms of arthritis, generated responses to OspA, LFA-1xcex1, and their highly related peptide epitopes. This finding indicates that the initial immune system response to infection with B. burgdorferi generates OspA-primed T cells which remain activated by stimulation with LFA-1xcex1 after the spirochete has been eliminated from the individual, leading to the release of inflammatory cytokines by these activated T cells and macrophages and resulting in tissue damage and joint destruction.
As prevention of tick infestation is imperfect, and Lyme disease may be missed or misdiagnosed when it does appear, there exists a continuing urgent need for an improved vaccine for the prevention of Lyme disease. Given the potential cross-reactivity between OspA and LFA-1a, the use of OspA as a protective immunogen in vaccines may be associated with the induction of an auto-immune reaction in certain populations, including individuals expressing the HLA-DRB1*0401 allele. Thus, it would be highly desirable to generate modified OspA polypeptides with diminished or no binding to the HLA-DRB1*0401 allele.
We have now discovered a modified OspA polypeptide which has diminished binding to HLA-DRB1*0401. The present invention provides a vaccine composition comprising an immunogenic amount of OspA, or a unique fragment of OspA, and a pharmaceutically acceptable carrier or vehicle, wherein said amount is sufficient to immunize a susceptible mammal against Lyme borreliosis, wherein said OspA has diminished binding to HLA-DRB1*0401.
A preferred embodiment of the invention provides a vaccine composition which includes the modified OspA polypeptide of SEQ ID NO:4, or any unique fragment of SEQ ID NO.:4.
Another embodiment of the invention provides a nucleotide sequence encoding a peptide as set forth in SEQ ID NO:4. The nucleotide sequence can further include a 5xe2x80x2-flanking region containing at least one promoter sequence for expression of the peptide.
Another embodiment of the invention provides a nucleotide sequence encodes a fusion polypeptide containing OspA, wherein said OspA has diminished binding to HLA-DRB1*0401 and comprises SEQ ID NO:4.
A further embodiment of invention provides a vector containing the isolated DNA molecule of SEQ ID NO:4.
A preferred method of the invention provides a method of protecting a susceptible mammal against Lyme borreliosis by administering an effective amount of a vaccine composition, in which the vaccine composition includes the modified OspA polypeptide of SEQ ID NO:4. Preferably, the vaccine composition is administered by subcutaneous or intramuscular administration. Even more preferably, the vaccine composition is administered orally.
The present invention also provides methods for producing a vaccine composition containing a substantially pure OspA polypeptide, by recovering the OspA polypeptide from a host organism transformed with a vector containing DNA encoding the OspA polypeptide, and admixing the OspA polypeptide with an immunologically acceptable carrier or vehicle.
Another embodiment of the invention provides a method for producing the vaccine composition by admixing the OspA polypeptide and the carrier or vehicle. Preferably, the method further includes adding an adjuvant.